Solid Phase Extraction (SPE) has rapidly evolved to become one of the most popular methods for sample preparation. SPE is widely used, for example, to isolate and/or concentrate organic analytes from sample matrices and to provide suitable sample extracts for instrumental analysis. Based on well-known chromatographic principles, SPE is relatively easy to perform and provides superior results compared to traditional liquid--liquid extraction methods.
The term "solid phase extraction" is used because the support material (sorbent bed packing) is a solid, through which a liquid is passed. Typically, a column tube or cartridge, or other support, holds the sorbent bed packing and the liquid phase carries the sample through it. Analytes are adsorbed and eluted according to their differential affinities between the sorbent material and the mobile phase. As the analytes elute, they can be quantified by a detector and/or collected for further analysis.
For the isolation of target compounds bearing non-polar functional groups, a non-polar sorbent is generally employed. According to this technique, referred to as reversed-phase SPE, target compounds are extracted from polar solvents or matrix environments due to hydrophobic interactions between the sorbent and target compounds as they pass through the sorbent bed. Elution of extracted compounds is achieved by organic-based solvents or solvent mixtures having sufficient non-polar character to disrupt the hydrophobic interactions.
In order for the sorbent in a reversed-phase SPE column to retain an analyte, it is necessary for a proper "phase interface" to exist between the sorbent and the sample. To this end, a solvation procedure is usually the first step of an extraction, wherein methanol, acetonitrile, or other organic solvent is passed though the column prior to application of a fluid sample. The solvent "wets" the solid phase, ensuring interaction of the analyte with the sorbent in the following sample application step. For example, with C.sub.18 -bonded silicas, solvation of the hydrocarbon chains allows the hydrophobic ligands to interact properly with organic analytes/solutes present in an aqueous sample. In the absence of such solvation, hydrophobic ligands interact strongly with each other rather than interacting effectively with the organic analytes in aqueous media, often resulting in poor recoveries of the organic analytes.
The general acceptance and perceived importance of sorbent conditioning in the field is underscored by the precautionary statements that users and manufacturers of conventional reversed-phase SPE sorbents often add to the descriptions of their experimental protocols and/or instructions for use. For example:
Statement: "Chemically bonded silica and porous polystyrene resins have several shortcomings for use in SPE. While silica itself is hydrophilic, the hydrocarbon chains make the surface hydrophobic. The consequence is poor surface contact with predominantly aqueous solutions. Porous polystyrene resins also have a hydrophobic surface. Pretreatment of the SPE materials with an activating solvent (such as methanol, acetone or acetonitrile) must be used to obtain better surface contact with the aqueous solution being extracted." PA1 Reference: p. 136 in "Methods and materials for solid-phase extraction," J. S. Fritz, P. J. Dumont, and L. W. Schmidt, J. Chromatogr. A, 691 (1995)133-140. PA1 Statement: "Cartridge conditioning is important for reversed-phase sorbents. Wetting the sorbent with an organic solvent such as methanol or acetonitrile will solvate the entire stationary phase and provides the maximum accessible surface for sample adsorption. After conditioning the sorbent with organic solvent, you should keep the bed properly wetted before sample introduction. Premature analyte breakthrough and subsequent variable recoveries can result from improper conditioning or failure to keep the sorbent wetted." PA1 Reference: p.856 in "SPE Method Development and Troubleshooting," E. S. P. Bouvier, LC-GC, 13(11) (1995) 852, 854, 856, 858. PA1 Instruction: "Solid Phase Extraction Procedure: . . . No step should be omitted . . . 4a. Condition: Add to and draw through each cartridge 1 mL methanol" (emphasis original). PA1 Reference: "Waters Oasis HLB Extraction Cartridges, Instruction Sheet," PIN WAT058812 Rev 0, 1996, Waters Corporation. PA1 a particle size of about 20-200 .mu.m, and preferably about 50-100 .mu.m; PA1 a surface area of about 300-1,000 m.sup.2 /g, and preferably about 450-650 m.sup.2 /g; PA1 a pore size range of about 10-800 .ANG., and preferably about 10-500 .ANG.; and PA1 a pore volume of about 1.0-1.5 ml/g, and preferably about 1.15-1.20 ml/g. PA1 a particle size of about 20-200 .mu.m, and preferably about 75-150 .mu.m; PA1 a surface area of about 300-1,000 m.sup.2 /g, and preferably about 450-550 m.sup.2 /g; PA1 a pore size range of about 10-800 .ANG., and preferably about 300-600 .ANG.; and PA1 a pore volume of about 1.0-1.5 ml/g, and preferably about 1.25-1.35 ml/g. PA1 R.sub.1 is an alkyl group having from 1 to 6 carbons, an alkoxy group having from 1 to 6 carbons, or a halogen group; PA1 R.sub.2 is an alkyl group having from 1 to 6 carbons, an alkoxy group having from 1 to 6 carbons, or a halogen group; PA1 R.sub.3 is an alkyl group having from 1 to 6 carbons, an alkoxy group having from 1 to 6 carbons, or a halogen group; PA1 m is an integer of from 1 to 8; and PA1 n is an integer of from 0 to 17. PA1 a particle size of about 50-100 .mu.m; PA1 a surface area of about 450-650 m.sup.2 /g; PA1 a pore size range of about 10-500 .ANG.; and PA1 a pore volume of about 1.15-1.20 ml/g. PA1 a particle size of about 75-150 .mu.m; PA1 a surface area of about 450-550 m.sup.2 /g; PA1 a pore size range of about 300-600 .ANG.; and PA1 a pore volume of about 1.25-1.35 ml/g. PA1 R.sub.1, R.sub.2, and R.sub.3 are, separately, an alkyl group having from 1 to 6 carbons, an alkoxy group having from 1 to 6 carbons, or a halogen group; PA1 m is an integer of from 1 to 8; and PA1 n is an integer of from 0 to 17. PA1 1) sample application (without conditioning/pretreatment); PA1 2) a wash with water or a mixture of water with low content of organic solvent (e.g., 5% methanol in water); and PA1 3) organic solvent elution.
Statements and instructions such as these reflect the "conventional wisdom" in the field that SPE sorbent conditioning is necessary in order to obtain acceptable results. In the absence of a conditioning step, it is generally accepted that a reversed-phase sorbent will not be able to properly interact with the organic analytes present in an aqueous sample and, as a result, the analytes will not be effectively retained.
The present invention is based in part on the discovery that certain SPE sorbents provide excellent recoveries for a wide range of organic compounds from aqueous solutions without any conditioning step. It should be appreciated that by eliminating the sorbent-conditioning step, the present invention permits faster and more efficient sample processing. This is significant since sample preparation is widely regarded as the primary bottleneck in the analysis of small organic compounds in aqueous fluids (e.g., drugs in biofluids).